Project 3: Role of Tumor Stem Cells in Recurrent Prostate Cancer The hypothesis of this project is that perturbation of the prostate tissue microenvironment by androgendeprivation therapy contributes to the transition of prostate cancer from androgen-stimulated to recurrent growth by affecting directly and indirectly multiple cell populations of the neoplastic prostate, resulting in alteration of: a) the pattern of differentiation of the progeny of prostate tumor stem cells;b) the vascularization of the prostate tissue microenvironment;and c) the number and role of neuroendocrine cells. The complex response of the prostate to castration will be investigated in model systems of mouse prostate cancer in a mouse tissue microenvironment (TRAMP), human prostate cancer in a mouse tissue microenvironment (CWR22) and human prostate cancer in a human tissue microenvironment (primary human xenografts of prostate tissue). The role of the prostate tissue microenvironment, and AR function, in progression of CaP in androgen-rich and androgen-deprived environments will be investigated in rat prostate tumor cell lines transplanted ectopically and orthotopically into syngeneic hosts of various ages. The experimental plan to test our hypothesis is developed in three specific aims. Specific Aim 1 will determine the impact of the tissue microenvironment on the differentiation of prostate stem cells/prostate tumor stem cells. These studies will focus on the identification, isolation and molecular characterization of human prostate stem cells from benign and malignant prostate specimens, the importance of AR in tumorigenic potential, and establishing the range of phenotypic plasticity that rat and human prostate stem cells and prostate tumor stem cells demonstrate in vitro and when transplanted in vivo to ectopic and orthotopic tissue microenvironments in syngeneic or immunocompromised hosts. Specific Aim 2 will determine if androgen deprivation induces hypoxia, vascular regression of immature vessels, VEGF upregulation, neovascularization/angiogenesis and epithelial cell involution in human prostate primary xenografts, in models of prostate cancer, and in human prostate cancer. These studies will focus on the androgen deprivation-induced perturbation of the molecular signals that determine vascular integrity and neovascularization in short-term human xenografts of benign and neoplastic prostate tissue, in the TRAMP model, in the CWR22 xenograft. Specific Aim 3 will determine whether neuroendocrine cells that develop in the prostate during androgen deprivation are the progeny of a prostate stem cell/tumor stem cell, or arise by "trans-differentiation" of prostatic adenocarcinoma cells, and characterize their function in the androgendeprived prostate. These cancer studies will focus on identification of the cells undergoing neuroendocrine differentiation in response to androgen deprivation in short-term human xenografts of benign and neoplastic prostate tissue, in the TRAMP model, and in the CWR22 xenograft model, and on characterization of the role of NE cells in initiation and maintenance of recurrent growth in these models.